Fcs express

pCEP4 plasmids containing wild type VN and VC fused receptors were used as templates for targeted mutagenesis by overlap extension PCR. Mutated inserts in all plasmids were confirmed by Sanger sequencing. Plasmids were transiently transfected as described above into the relevant cell line and analyzed by flow cytometry for receptor expression and BiFC signal 24 h post-transfection. Cells were washed with PBS-BSA, incubated in 1:100 anti-myc Alexa 647 and 1:100 anti-FLAG Cy3 in PBS-BSA for 20 minutes, washed twice, and resuspended in PBS-BSA for analysis on a BD LSR II. Data were collected using instrument software and analyzed with FCS Express (De Novo Software). Cells were gated by forward-side scatter for the main population, and then gated by Alexa 647/Cy3 fluorescence to control for a consistent level of surface expressed receptor across the samples. Mean YFP/Venus fluorescence was recorded.

Peripheral blood mononuclear cells (PBMC) from a healthy control were isolated with density gradient centrifugation and aliquoted by 500 000 cells to test tubes containing IFN-α2a (10 000 U/ml) pre-incubated with serum dilutions for 2 hr. Tubes with or without IFN alone served as positive and negative controls. After 15 min of stimulation of PBMCs at 37°C, the cells were fixed immediately with Cytofix buffer, permeabilized with Perm Buffer III and stained with PE-conjugated antibody to phospho-STAT1 (Y701; all from BD Biosciences). Data were acquired with LSRFortessa (BD Biosciences) and analyzed with FCS Express (De Novo Software).

Flow cytometric analysis of pSTAT1 [14 (link)] and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis [16 (link)] of nitrated STAT1 (nSTAT1) were conducted on all samples. For flow cytometry studies, frozen PBMCs were thawed at 37 °C, washed with culturing media, and allowed to rest overnight in complete media at 5% CO₂ at 37 °C. PBMCs were stimulated with interferon-α (Miltenyi Biotec, Cambridge, MA, USA) at 0, 10² U/mL, and 10⁴ U/mL and incubated for 15 min in media, as previously described [10 (link)].
The live/dead marker Zombie NIR (BioLegend, San Diego, CA, USA) was used before permeabilization to distinguish live cells. The samples were permeabilized using the FIX & PERM Cell Permeabilization Kit with methanol modification (Fisher Scientific, Hampton, MA) and fixed at −20 °C for a minimum of 2 h. pSTAT1 was detected by a pSTAT1 antibody (AF488; BD Biosciences, San Jose, CA, USA). Flow cytometry data were collected either on Canto or LSRII flow cytometers (BD Biosciences, San Jose, CA, USA), and data were analyzed in FCS Express (De Novo Software, Pasadena, CA, USA). Measurement of patient-derived PBMCs for nSTAT1 and native STAT1 via LC-MS-MS SRM experiments was performed as described previously [16 (link)].

To determine their viability, T cells were stained with Annexin V dye conjugate to determine apoptotic cells and ToPro3 (Thermo Fisher Scientific) to determine dead cells. T cells were re-suspended in dyes as recommended by the manufacturer and incubated for 20 min on ice. Cells were washed and centrifuged at 350 x g for 8 min and analysed by flow cytometry on a LSRII (BD Biosciences) using Diva Software. Analysis of flow cytometry data was performed using FCS Express (De Novo).

Cells were washed with MACS flow buffer (MACS, 130-091-222) and permeabilized with BD Cytofix/Cytoperm (BD, 554714) prior to incubation with antibodies. Cells were labeled for antibodies against CD90 (Abcam, ab3105; Abcam, ab124527; Abcam, ab23894; BD, 555595), CD105 (Abcam, ab107595; Abcam, ab2529; Abcam, ab11414; R&D Systems, Fab10971p), Pro-SPC (Bioss, bs 10067R; Abcam, ab40879), CCSP (Abcam, ab171957), Epcam (Abcam, ab71916, Abcam, ab168828; Life Technologies, a15755), and Aqp5 (Abcam, ab78486; Abcam, ab85905) and detected by Alexa Fluor 488 (Abcam, ab150117, ab150077) or fluorescein isothiocyanate (FitC) (Abcam, ab6840) secondary antibodies. Both unstained and isotype controls (Abcam, ab18419; BD, 559320; Abcam, ab125938) were utilized as controls. Human adipose-derived mesenchymal stem cells (AD-MSCs) were obtained from Lonza. Flow Cytometry was performed on the CytoFlex (Beckman Coulter, Indianapolis, IN) and analyzed using FCS Express (De Novo Software, Glendale, CA) or CytExpert ((Beckman Coulter, Indianapolis, IN).

Antibodies against human CD3 (BW264/56) and CD45RA (T6D11) were purchased from Miltenyi Biotec. Antibodies against human CD3 (UCHT1), CD25 (BC96), CD127 (A019D5), PD-1 (EH12.2H7), CCR7 (G043H7), and CD155 (SKII.4) were obtained from Biolegend. Fixable viability dye and antibodies against human CD4 (RPA-T4), CD8α (RPA-T8), and Foxp3 (PCH101) were purchased from Thermo Fisher Scientific, and antibody against human CD96v2 (628211) was obtained from R&D Systems. Fluorescently labeled F(ab′)2 against FcγRI (10.1), FcγRIIA/B (AT10), and FcγRIIIA (3G8) were provided by Mark Cragg, University of Southampton. FcγR staining was performed in PBS/1% BSA, without a prior FcγR blocking step. For CFSE proliferation assays and analysis of CD96 and CD155 expression, cells were incubated for 10 minutes at 4°C with 10% heat-inactivated AB serum (MilliporeSigma) prior to surface staining. For TIL phenotyping, cells were incubated with human FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4°C prior to surface staining. When required, intracellular staining was performed using the Foxp3 staining buffer kit (Thermo Fisher Scientific). Samples were analyzed with a FACSCanto II flow cytometer and DIVA Software (BD Biosciences), FCS Express (De Novo Software), or FlowJo software (version 10).